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Mitochondrial-Associated Protein LRPPRC Is Related with Poor Mitochondrial-Associated Protein LRPPRC is Related With Poor Prognosis Potentially and Exerts as an Oncogene via Maintaining Mitochondrial Function in Pancreatic Cancer Qiongying Hu Department of Laboratory Medicine, hospital of chengdu university of traditional chinese medicine Li Wang Department of Pancreatic Surgery, west china hospital of sichuan university Yi Zhang Department of pancreatic surgery, west china hospital of sichuan university Bole Tian Department of pancreatic surgery, west china hospital of sichuan university ziyi Zhao ( [email protected] ) Hospital of Chengdu University of Traditional Chinese Medicine https://orcid.org/0000-0003-1871- 5197 Ya Zhang Department of Endocrinology, hospital of chengdu university of Traditional chinese medicine Primary research Keywords: LRPPRC, autophagy/mitophagy, pancreatic cancer, mitochondrial homeostasis, chemoresistance, reactive oxygen species (ROS) Posted Date: February 8th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-171632/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/23 Abstract Background The mitochondrial-associated protein LRPPRC exerts multiple functions involved in physiological processes, including mitochondrial gene translation, cell cycle progression and tumorigenesis. Previously, LRPPRC was reported to regulate mitophagy by interacting with Bcl-2 and Beclin 1 and thus modifying the activation of PI3KCIII and autophagy. Considering that LRPPRC was found to be negatively associated with survival rate, we hypothesize that LRPPRC may be involved in pancreatic cancer progression via its regulation of autophagy. Methods real-time quantitative PCR was performed to detect the expression of LRPPRC in 90 paired pancreatic cancer and adjacent tissues and ve pancreatic cancer cell lines. Mitochondrial reactive oxidative species (ROS) level and function were measured. Mitophagy was measured by performing to detect LC3 level. Results By performing RT-qPCR, the association of LRPPRC with the prognosis of pancreatic cancer was established and pancreatic cancer tissues had signicantly higher LRPPRC expression than adjacent tissues. LRPPRC was negatively associated with the overall survival rate. LRPPRC was also upregulated in pancreatic cancer cell lines. Knockdown of LRPPRC promoted ROS accumulation, decreased mitochondrial membrane potential (MMP), promoted autophagy/mitophagy, and induced mitochondrial dysfunction. Subsequently, knockdown of LRPPRC inhibited malignant behaviors in PANC-1 cells, including proliferation, migration, invasion, tumor formation and chemoresistance to gemcitabine. Finally, by inhibiting autophagy/mitophagy using 3-MA, the inhibitory effect of LRPPRC knockdown on proliferation was reversed. Conclusion Taken together, our results indicate that LRPPRC may act as an oncogene via maintaining mitochondrial homoeostasis and could be used as a predictive marker for patient prognosis in pancreatic cancer. Introduction Pancreatic cancer is one of the leading causes of cancer-related death and has a very low overall survival rate of approximately 5% and a median survival of less than 6 months [1]. In patients suffering from therapy failure, including induction of chemoresistance and radioresistance, distant metastasis before surgical operation has been discovered [2]. Pancreatic cancer is characterized by diverse mutations, including RAS, Smad4 and p53, and thus frequently exhibits chemoresistance [3] and insensitivity to radiotherapy [4]. Our earlier study revealed that pancreatic cancer and cancer stem-like cells (CSCs) derived from pancreatic cancer cells have higher levels of long noncoding RNAs and thus induce Page 2/23 chemoresistance [5]. Even so, we still know little about the regulators of malignant behaviors of pancreatic cancer. Leucine-rich pentatricopeptide repeat-containing (LRPPRC), also known as LRP130, is a member of the pentatricopeptide repeat protein family and exerts multiple functions involving homeostasis, microtubule alterations, RNA stability, DNA/RNA binding, transcriptional activity in mitochondria, metabolic processes, RNA nuclear export, tumorigenesis and tumor progression [6–10]. LRPPRC is localized to both the cytoplasm and mitochondria [8]. In the mitochondrial matrix, LRPPRC binds to single-stranded RNA and thus posttranscriptionally regulates mitochondrial genes and subsequently regulates mitochondrial functions [11]. In cancer progression, LRPPRC was also reported to be associated with mitochondria by interacting with Parkin and thus stabilizing Parkin substrates, including Bcl-2 and Parkin itself to inhibit autophagy, and consequently, LRPPRC protects mitochondria from autophagy degradation [12]. Knockdown of LRPPRC causes a decrease in Bcl-2, followed by Beclin 1 release to form complexes with PI3KCIII to activate basal levels of autophagy [13]. In this manner, LRPPRC acts as an autophagy/mitophagy inhibitor via maintaining mitochondrial membrane potential (MMP) and thus promoting mitochondrial function (14). Mitochondria play essential roles in physiological processes, including energy production, cell signaling and apoptosis [15, 16]. Reactive oxygen species (ROS), as a byproduct of energy production in mitochondria, have been shown to promote protein oxidation and consequent misfolding and/or unfolding of mitochondrial proteins localized in the mitochondrial matrix [17, 18]. Under physiological conditions, the balance between ROS accumulation and ROS scavenging is strictly regulated to avoid oxidative stress, which can regulate the mitochondrial permeability transition pore (mPTP) and thus maintain healthy mitochondrial homeostasis [13]. By considering that LRPPRC is reported to exert critical roles in maintaining MMP via regulating autophagy/mitophagy, it is supposed that LRPPRC might regulate ROS accumulation and scavenge and thus maintaining MMP. Previous reports have shown that LRPPRC may act as an oncogene in several kinds of cancer, including hepatoma, lung adenocarcinoma, esophageal squamous cell carcinoma and colon cancer [19]; however, the exact role of LRPPRC in pancreatic cancer and whether its regulatory effect on mitochondria is involved in these processes are still unclear. In this study, we detected the expression level of LRPPRC in 90 pancreatic cancer and paired adjacent tissues and ve pancreatic cancer cell lines to investigate the relevance of LRPPRC expression. Furthermore, we also established the potential association of its regulation of mitochondrial function with the malignant behaviors of pancreatic cancer, especially cell proliferation. In conclusion, our results indicated that LRPPRC may be a critical marker for prognosis and a potential therapeutic target in pancreatic cancer. Material And Methods Clinical tissue samples Page 3/23 Our study included 90 patients (57 males, 33 females; mean age 61.9 years; range 36–85), were recruited from May 2010 and August 2018 after obtaining their writtern informed consent. All patients survived from 3 to 7.2 years were conrmed by telephone and mail. The study items included age, gender, tumor size, and tumor-node-metastasis stage. Patient characteristics are summarized in Table 1. Tissues were xed in 10% formaldehyde, embedded in paran, cut into 1.5 mm in diameter and 4 µm in thick, and mounted on a tissue microarray. Table 1 Correlation between clinicopathological characteristics and LRPPRC expression Total n LRPPRC P low high Age 0.361 ≤60 40 15 25 60 50 20 30 Gender 0.219 Male 57 21 36 Female 33 11 22 Tumor size 0.195 ≤5cm 55 21 34 5cm 35 12 23 N 0.016* N0 51 21 30 N1 39 12 27 * P value for expression levels compared by Mann-Whitney test Immunohistochemical staining The tissue microassay were stained for immunohistochemical analysis. Microarray was baked at 60℃ for 2 h, deparanization with xylene, and then rehydrated, after being washed three times in 1×PBS. Then, rehydrated microarray was incubated with 3% hydrogen peroxide for 10 min in methanol to inactivate endogenous peroxidase activity, and then blocked using 2.5% bovine serum albumin (BSA) dissolved in PBS against nonspecic binding sites for 30 min at room temperature (RT). Then anti- LRPPRC antibody (diluted in 1:200; Cat. No.: ab97505; Abcam, Cambridge, England) was added for over- night incubation at 4℃. Incubated microarray was then rinsed for three times in ice-cold PBS and Page 4/23 incubate with a horseradish-peroxidase-conjugated antibody (diluted in 1:5000; Cat. No.: ab7090; Abcam) for 1h at RT. The microarray was developed then with 3, 3’-diaminobenzidine solution for 2–5 min, washed briey in running water and imaged under a microscope (Olympus BX51; Olympus, Japan). Immunohistochemical analysis Staining of LRPPRC was mainly detected in the cytoplasm of tumor tissues, and slightly observed in the nucleus of tumor tissues. The ratio of positive stained cells, intensity of stained cells and staining score were reviewed independently by two pathologists without knowing the clinical features or survival status of the patients. The ratio of positive stained cells was graded as follows: 0 = staining of ≤ 1 %; 1 = staining of 1–20%; 2 = staining of 21–40%; 3 = staining of 41–60%; 4 = staining of 61–80% and 5 = staining of ≥ 81%. The intensity of stained cells was graded as follows: 0 = no signal; 1 = week signal; 2 = moderate signal; 3 = strong signal. The staining score was graded as follows: The ratio of positive stained was graded as follows: low expressing group = the ratio of positive stained
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